Investigating the role of the ETS transcription factor ELK1 in stem cell transcription A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2017 Ian E. Prise Division of Molecular & Cellular Function School of Biological Sciences I. Table of Contents II. List of Figures ...................................................................................................................................... 5 III. Abstract .............................................................................................................................................. 7 IV. Declaration ......................................................................................................................................... 8 V. Copyright Statement ........................................................................................................................... 8 VI. Experimental Contributions ............................................................................................................... 9 VII. Acknowledgments .......................................................................................................................... 10 1. Introduction ...................................................................................................................................... 12 1.I Pluripotency ................................................................................................................................. 12 1.II Chromatin ................................................................................................................................... 13 1.II.A Histones ............................................................................................................................... 14 1.III Transcription .............................................................................................................................. 15 1.III.A Transcriptional activation ................................................................................................... 15 1.III.B Transcriptional repression .................................................................................................. 17 1.III.C Polycomb repression complexes ........................................................................................ 20 1.IV The mitogen activated protein kinase pathways....................................................................... 24 1.IV.A Response to different stimuli ............................................................................................. 24 1.V The ETS transcription factor family............................................................................................. 25 1.VI ELK1............................................................................................................................................ 26 1.VI.A Structure of ELK1 ................................................................................................................ 26 1.VI.B Relationship to other ETS proteins ..................................................................................... 29 1.VI.C Biological processes involving ELK1 .................................................................................... 30 2. Project aims....................................................................................................................................... 32 3. Materials and Methods ..................................................................................................................... 35 3.I Lab techniques ............................................................................................................................. 35 3.I.A Cell Culture ........................................................................................................................... 35 3.I.B Retinoic acid treatment ........................................................................................................ 35 3.I.C Mesoderm Differentiation .................................................................................................... 36 3.I.D Neural Progenitor Cell Differentiation.................................................................................. 36 3.I.E IL-6 Cell stimulation ............................................................................................................... 36 3.I.F shRNA treatment ................................................................................................................... 36 3.I.G siRNA transfection ................................................................................................................ 36 3.I.H Crosslinking and lysing cells .................................................................................................. 37 3.I.I Chromatin Immunoprecipitation ........................................................................................... 37 3.I.J Co-Immunoprecipitation ....................................................................................................... 38 3.I.K RNA purification .................................................................................................................... 38 3.I.L PCR (siELK1, MIM and NPC treatments) ................................................................................ 38 3.I.M RT-PCR (shELK1 and RA treatments) ................................................................................... 39 3.I.N Rapid immunoprecipitation of endogenous proteins .......................................................... 39 3.I.O Western Blot Analysis ........................................................................................................... 40 3.II Bioinformatic analysis ................................................................................................................. 40 3.II.A ChIP-seq analysis.................................................................................................................. 40 3.II.B RNA-seq analysis .................................................................................................................. 41 3.II.C Peak Intersections ................................................................................................................ 41 3.II.D Tag Density Graphs .............................................................................................................. 41 2 3.II.E Motif Analysis ....................................................................................................................... 41 3.II.F Graphics ................................................................................................................................ 42 3.II.G Accession numbers .............................................................................................................. 42 3.II.H Primer Design ...................................................................................................................... 42 3.II.I Statistical Analysis ................................................................................................................. 42 3.III Materials .................................................................................................................................... 42 3.III.A siRNAs ................................................................................................................................. 42 3.III.B PCR Primers ......................................................................................................................... 42 3.III.C ChIP Solutions ..................................................................................................................... 46 3.III.D Western Blot Solutions ....................................................................................................... 48 3.III.E Antibodies ........................................................................................................................... 48 4. Results ............................................................................................................................................... 50 4.I ELK1 binding regions can be divided into different modules ...................................................... 50 4.I.A Genomic distribution of ELK1 modules ................................................................................ 50 4.I.B ELK1 loci with low ERK2 enrichment correlate with high PRC2 enrichment ........................ 52 4.I.C ELK1 loci with high SUZ12 enrichment correlate with low enrichment of active histone modifications ................................................................................................................... 54 4.I.D ELK1 loci with high SUZ12 enrichment correlate with genes enriched for developmental GO terms .............................................................................................................. 56 4.I.E An ELK1+SUZ12 binding module is H1-hESC specific ............................................................ 58 4.I.F ELK1 loci with low GABPA enrichment correlate with high PRC2 enrichment ..................... 60 4.I.G Conclusion Part1 ................................................................................................................... 65 4.II ELK1 does not interact with PRC2 ............................................................................................... 67 4.II.A An